Process for diagnosis of neurodegenerative diseases

ABSTRACT

The invention provides an analytical process for analysing the presence of at least one aggregated conformation prion protein in a sample of body fluid or a sample of tissue and uses the dependency of the amplification of the aggregated conformation on the shear-force intensity applied to the native conformation prion protein, which is also dependent on the specific seed present in the admixture with native conformation prion protein, for specifically analysing for the presence of an aggregated conformation prion protein in the sample. The process of the invention contains the step of determining the content of aggregated conformation prion protein generated in admixture with the sample to be analysed using one shear-force intensity, preferably using least at two different shear-force intensities and the step of comparing data on these contents of generated prion protein having an aggregated conformation with data on the content of aggregated prion protein that is pre-determined, each at the same shear-force intensity for a mixture of the same native conformation prion protein with a reference sample as a seed.

The present invention relates to an analytical process for use in thediagnosis and classification of neurodegenerative diseases, especiallyneurodegenerative diseases associated with protein misfolding or proteinaggregation, and to a device for use in the process. Herein,neurodegenerative diseases associated with protein misfolding are alsocollectively referred to as prion-aggregate related diseases, includinge.g. amyloidoses, and proteopathies that include synuclein aggregationdiseases (synucleopathies), tau aggregation diseases (tauopathies) andAlzheimer beta (Aβ) peptide aggregation diseases. Preferred diseases areneurodegenerative disorders and dementias, e.g. Alzheimer's disease,frontotemporal lobular dementia, Parkinson disease, Dementia with LewyBodies, Parkinson's Disease with Dementia, Multiple System Atrophy,amyotrophic lateral sclerosis, motor neuron disease and Huntingtondisease. These diseases are accompanied by occurrence of aggregatedconformation prion protein which presents a suitable pathologicalanalyte having specificity for such a disease. The aggregatedconformation prion protein can be transmissible and is able to inducenative conformation prion protein to change its conformation to theaggregated conformation associated with disease pathology. Further, theinvention relates to a device for use in the process.

STATE OF THE ART

Salvadores et al., Cell Reports 261-268 (2014) describe the detection ofaggregated Amyloid-β (Aβ) protein for diagnosis of Alzheimer's diseaseby incubating in a temperature-controlled shaker a cerebrospinal fluid(CSF) sample with aggregate-free Aβ peptide and Thioflavin T. Amyloidformation was determined by intermediate fluorometry using a platespectrofluorometer for Thioflavin T binding to amyloid fibrils.

WO 2012/110570 A1 describes a process for the amplification ofaggregated conformation prion protein from native conformation prionprotein in admixture with aggregated conformation prion protein usingshear-force control. Optionally, aliquots of an admixture are subjectedto different intensities of controlled shear-force. The disclosure of WO2012/110570 is contained herein by reference.

Eckroat et al., Beilstein J. Org. Chem. 2013, 1012-1044 describe dyeprobes for Amyloid-ß. McKhann et al, Neurology. 34(7), 939-44 (1984),Jack Jr et al, Alzheimers Dement. 2011 7(3), 257-262, McKhann et al.,Alzheimers Dement. 7(3): 263-269 (2011), Albert et al., AlzheimersDement. 7(3): 270-279 (2011), Sperling et al., Alzheimers Dement. 7(3):280-292 (2011) and Jack Jr et al., Ann. Neurol. 71(6): 765-774 (2012) aswell as Eggert K, et al. “Leitlinien: Parkinson-Syndrome—Diagnostik undTherapie”, AWMF-Register Nr. 030/010, Stand 09/2012, and DeutscheGesellschaft für Psychiatrie, Psychotherapie und Nervenheilkunde(DGPPN), “S3 Praxisleitlinie: Diagnose- und Behandlungsleitlinie‘Demenz”’ describe diagnostic criteria for classifying neurodegenerativediseases. Barghorn et al., Methods in Molecular Biology, Vol 299, 2005:Amyloid Proteins: Methods and Protocols, pages 35-51, “Purification ofrecombinant tau protein and preparation of Alzheimer-paired helicalfilaments in vitro” describe isoforms of human tau protein.

OBJECT OF THE INVENTION

It is an object of the invention to provide an analytical process thatallows to detect an analyte specific for a prion-aggregate relateddisease using a biopsied sample obtained from a mammal, especially ahuman or an experimental mammal, e.g. a mouse or rat. A preferred objectis an analytical process that allows for a differential diagnosis, morepreferably prior to onset or early at onset of clinical symptoms of thedisease.

DESCRIPTION OF THE INVENTION

The invention attains the object, especially the differential diagnosisof a prion-aggregate related disease by the features of the claims,especially by an analytical process for analysing for the presence of atleast one aggregated conformation prion protein in a sample of bodyfluid or in a sample of tissue originating from a mammal, preferablybiopsied from a human, especially of a preclinical patient or a patientsuspected of having a neurodegenerative disease, e.g. a patientsuspected of having a neurodegenerative disease on the basis ofphenomenological diagnostic criteria as described in the state of art.The sample can comprise or consist of blood, preferably blood serum,urine and/or cerebrospinal fluid (CSF), or any other solid tissue, e.g.a tissue sample that preferably contains a nerve section, for example askin biopsy. The mammal preferably is a human, a farm animal used forfood production, e.g. a bovine, deer, elk, swine, sheep, goat, fowl, oran experimental animal, e.g. a mouse, rat or non-human primate. Theanimal can also be a wild mammal, for example wild goat, deer or elk.Herein, the generation of aggregated conformation prion protein from anadmixture comprising native conformation prion protein and a samplecontaining aggregated conformation prion protein with application ofshear-force is also referred to as amplification.

For the purposes of the invention, neurodegenerative diseases alsoinclude posttraumatic stress disorder (PTSD, e.g. ICD10 F43.1) that maydevelop in persons after being exposed to one or more traumatic events,e.g. sexual assault, fighting in a war, sustaining serious injury orthreat of death with the experience of intense fear, horror, orpowerlessness, as well as restless leg syndrome, (RLS, e.g. G25.8), alsoknown as Willis-Ekbom disease (WED) of Wittmaack-Ekbom syndrome, aneurological disorder characterized by an irresistible urge to moveone's body to stop uncomfortable or odd sensations, e.g. affecting thelegs, arms, torso, head or even phantom limbs, the movement providingtemporary relief.

During the preparation of the invention it has been found thataggregated conformation prion protein induces the change in conformationof native prion protein in dependence on the shear-force intensityapplied to a mixture of these, and that the change in conformation canin addition depend on the specific aggregated conformation prion proteinadded to the native conformation prion protein. As used herein, nativeprion protein relates to the non-aggregated conformation protein thatundergoes a change in conformation in the presence of aggregatedconformation prion protein (seed) to aggregated conformation prionprotein. Accordingly, native prion protein can also be referred to asnon-aggregated prion protein. In greater detail, it has been found thatdepending on the source, the aggregated conformation prion protein whensubjected to specific shear-force in admixture with native conformationprion protein can yield amplification of an aggregated conformation onlyfor specific shear-force intensities, whereas little or no amplificationof the aggregated conformation occurs at different shear-forceintensities, e.g. different shear-force intensities yield a differentpattern of aggregated conformation generated from the original nativeconformation prion protein. The aggregated conformation prion protein isalso termed proteopathic seed, or seed. For example, the seed and thenative conformation prion protein in the mixture subjected to differentshear force intensities can have the same amino acid sequence, resultingin the amplification of aggregated conformation prion protein from thenative conformation prion protein only at specific shear-forceintensities. Accordingly, the invention uses the dependency of theamplification of the aggregated conformation on the shear-forceintensity applied to the native conformation prion protein, which isalso dependent on the specific seed present in the admixture with nativeconformation prion protein, for specifically analysing for the presenceof an aggregated conformation prion protein in the sample. Accordingly,the process of the invention contains the step of determining thecontent of aggregated conformation prion protein generated in admixturewith the sample to be analysed using one shear-force intensity,preferably using least at two different shear-force intensities and thestep of comparing data on these contents of generated prion proteinhaving an aggregated conformation with data on the content of aggregatedprion protein that is pre-determined, each at the same shear-forceintensity for a mixture of the same native conformation prion proteinwith a reference sample as a seed. The at least two differentshear-force intensities can e.g. be at least 3, preferably at least 4 toat least 12, e.g. up to 24 or up to 36 different shear-forceintensities. Generally preferred, the shear-force intensity of theprocess is pre-determined and identical for the sample and for use inthe generation of pre-determined data on the content of aggregatedconformation prion protein, especially generated from an admixture ofnative conformation prion protein and a reference sample. Theshear-force intensity can generally be pre-determined in respect ofduration of shear-force application for each cycle, duration of restingphase for each cycle, and number of repetition of cycles.

Preferably, the pre-determined data on the content of aggregatedconformation prion protein generated is determined separately for eachof at least two mixtures of the same native conformation prion proteinwith a seed, each mixture containing a different reference sample as aseed, which is e.g. obtained from a different reference patient having aknown diagnosis for a neurodegenerative disease, for example determinedby conventional diagnostic schemes. Convential diagnostic schemes arediagnostic guidelines for dementia and Parkinson syndromes, e.g. theclassification according to McKhann et al, “Clinical diagnosis ofAlzheimer's diesease: report of the NINCDS-ADRDA Work Group under theauspices of Department of Health and Human Services Task Force onAlzheimer's Disease, Neurology. 34(7), 939-44 (1984), preferablyaccording to the overview by Jack Jr et al., “Introduction to revisedcriteria for the diagnosis of Alzheimer's disease: National Institute onaging and the Alzheimer association workgroups, Alzheimers Dement. 20117(3), 257-262, in respect of AD more preferred according to McKhann etal., “The diagnosis of dementia due to Alzheimer's disease:Recommendations from the National Institute on Aging—Alzheimer'sAssociation workgroups on diagnostic guidelines for Alzheimer'sdisease”, Alzheimers Dement. 7(3): 263-269 (2011), in respect of mildcognitive impairment (MCI) due to AD preferably according to Albert etal., “The diagnosis of mild cognitive impairment due to Alzheimer'sdisease: Recommendations from the National Institute onAging—Alzheimer's Association workgroups on diagnostic guidelines forAlzheimer's disease”, Alzheimers Dement. 7(3): 270-279 (2011), inrespect of preclinical AD preferably according to Sperling et al.,“Towards defining the preclinical stages of Alzheimer's disease:Recommendations from the National Institute on Aging—Alzheimer'sAssociation workgroups on diagnostic guidelines for Alzheimer'sdisease”, Alzheimers Dement. 7(3): 280-292 (2011) and/or according toJack Jr et al., “An operational approach to NIA-AA criteria forpreclinical Alzheimer's disease”, Ann. Neurol. 71(6): 765-774 (2012),and/or according to Eggert K, et al. “Leitlinien:Parkinson-Syndrome—Diagnostik und Therapie”, AWMF-Register Nr. 030/010,Stand 09/2012, and/or according to Deutsche Gesellschaft fürPsychiatrie, Psychotherapie und Nervenheilkunde (DGPPN), “S3Praxisleitlinie: Diagnose—und Behandlungsleitlinie ‘Demenz’”.Preferably, data on these pre-determined amounts which are specific forat least one shear-force intensity, preferably for at least twodifferent shear-force intensities and specific for a reference samplefrom a specific source are contained and provided in a computer-baseddatabank, and the comparison is carried out by a computer.

The specific reference sample can for example be selected from a sampleof body fluid or tissue of a patient, alive or deceased, with adiagnosis for a specific neurodegenerative disease or a subtype thereof.Preferably, the databank contains the diagnosis for a specificneurodegenerative disease and/or a subtype thereof in association withpre-determined data on amounts of aggregated conformation prion protein,each generated at one of at least two, e.g. 3, 4, 12, 24, or 36different shear-force intensities for each reference sample. Generally,the sample to be analysed and the reference sample can be admixed withthe same native conformation prion protein and subjected to the sameshear-force intensities.

The shear-force dependent amplification of the aggregated conformationis made by subjecting the mixture comprising or consisting of the sampleof body fluid and at least one native conformation prion protein to atleast two different shear-force intensities. The shear-force intensitiesare controlled to have a uniform intensity having an intensity range ofmaximally 20% or maximally 15% or 12%, e.g. maximally 10%, preferablymaximally 5%, more preferably maximally 2% or maximally 1% of oneshear-force value.

In a first preferred embodiment, the mixture comprising the mammaliansample, e.g. of body fluid and/or tissue, and at least one nativeconformation prion protein is subjected to at least two differentshear-force intensities by dividing the mixture into aliquots (portionsof the mixture) and subjecting each of the aliquots to one of the atleast two shear-force intensities. Preferably, the aliquots aresubjected to one of the shear-force intensities each in parallel,preferably using identical devices or identical sections of devices forgenerating the shear-force intensities.

In a second preferred embodiment, the mixture or an aliquot thereof issubjected to at least two different shear-force intensitiessequentially, e.g. subjected firstly to one of the shear-forceintensities and later to another of the shear-force intensities. In thisembodiment, the determination of the content of aggregated conformationis made during and/or subsequent to application of one shear-forceintensity and/or subsequent to application of each shear-forceintensity.

The first and second embodiments can be combined, e.g. to a process inwhich at least two aliquots of the mixture are each subjectedsequentially to different shear-force intensities, e.g. the mixture orat least two aliquots thereof are subjected to one first shear-forceintensity and subsequently to a least one second shear-force intensity,each shear-force intensity differing from the other.

In the process, native conformation prion protein is contacted withaggregated conformation prion protein (seed) in a liquid composition andsubjected to at least one cycle or to a number of cycles of applicationof shear-force for fragmenting aggregates of prion protein, wherein theshear-force applied is precisely controlled, e.g. to a range ofmaximally 20% or maximally 15% or 12%, e.g. maximally 10%, preferablymaximally 5%, more preferably maximally 2% or maximally 1% of oneshear-force value, more preferably to 0.5% intensity range around oneshear-force intensity value, wherein optionally each cycle contains atleast one second phase of incubation without agitation and/or at leastone phase of agitation at one shear-force intensity value, which isdifferent from the first shear-force intensity value, which is e.g. zeroor 1 to 50%, preferably zero of the first shear-force intensity value.The second phase of incubation, also referred to as a resting phase, isincluded for allowing the aggregation of native conformation prionprotein with aggregated conformation prion protein. Optionally, theproduct obtained by the process of the invention can be used asaggregated conformation protein, e.g. as seed for pre-determination ofthe content of aggregated state prion protein. For the pre-determinationthe seed in admixture with native conformation prion protein issubjected to at least one cycle or to a number of cycles of applicationof shear-force for fragmenting aggregates of prion protein as describedherein.

Native conformation prion protein can e.g. be produced by expression ina cultivated cell which is genetically manipulated to contain anexpression cassette encoding the prion protein and isolating the prionprotein from the cultivated cell and/or from the medium of a culture ofthe cells. Cells for expression of prion protein can be bacteria,preferably E. coli, yeast, fungi, and mammalian cells, e.g. human cellsor hamster CHO cells. Native conformation prion protein can also beproduced from mammalian tissue.

The shear-force intensities can be generated as cycles or repetitions ofa fragmentation phase with a subsequent resting phase withoutshear-force being applied, e.g. each fragmentation phase consisting of 1s to 240 s, preferably 30 s to 120 s and each subsequent resting phaseconsisting of 10 s to 1080 s, preferably 60 s to 540 s. The cycles orrepetitions of the fragmentation phase and the subsequent resting phasecan e.g. be from 1 to 500 cycles, e.g. 5 to 500, preferably 60 to 280cycles, e.g. 100 to 140 cycles. In each cycle, the shear-force iscontrolled to be within an intensity range of maximally 10%, preferablymaximally 2%, more preferrably maximally 1%, e.g. within 0.5% or within0.1% of one shear-force intensity.

The shear-force having an intensity that is controlled to have a uniformintensity having an intensity range of maximally 20% of one shear-forcevalue can be generated by a shear-force generator comprising orconsisting of a rotary element arranged in a sample vessel having a lid,wherein the rotary element is run on bearings which are coaxiallyarranged within the sample vessel, and wherein the rotary elementcomprises a first coupling element of a coupling, e.g. comprising orconsisting of a permanent magnet, which preferably is at least bipolaror quadrupolar. The first coupling element is arrangeable for couplingwith the second coupling element of the coupling, e.g. a driving coilarrangement which can be arranged surrounding the first couplingelement. Preferably, the outer surface of the rotary element is parallelto the inner wall surface of the vessel, e.g. the rotary element and acoaxial section of the inner wall surface of the vessel are spaced andcylindrical or conical. Preferably, the bearing of the rotary elementcomprises or consists of an axle, one end of which is arrangedcontacting a bottom section of the inner vessel surface and the otherend of which runs in a bearing attached next to the rim of the vessel,e.g. by frictional connection and/or by positive fit.

Alternatively, the shear-force can be generated by a shear-forcegenerator having a rotary element coaxially arranged in a radiallyspaced tube section, the radial spacing of the rotary element and thetube and the axial section in which both the rotary element and the tubeextend defining a space, e.g. of ring-shaped cross-section, in whichspace upon rotation of the rotary element shear-force is generated.Preferably, the rotary element along its longitudinal and rotary axishas a constant outer diameter and is arranged at a constant spacing fromthe encircling tube section. The rotary element can take any outer form,preferably of axial symmetry, e.g. a flat shape or a rectangularcross-section and preferably has a cylindrical outer surface.Preferably, the tube in its section encircling the rotary element has acylindrical inner cross-section. Along the common longitudinal axis, thetube preferably at one end of the section encircling the rotary elementextends over the rotary element, such that the rotary element ends at adistance from the end of the tube, allowing a suction action at rotationof the rotary element, and at the opposite end of the section encirclingthe rotary element, the rotary element extends over at least one exitopening in the walls of the tube, allowing liquid to exit. Preferably,the at least one exit opening in the walls of the tube has across-section of at least the cross-section of the area between the tubeand the rotary element, more preferably, the exit opening has across-section of or greater than the inner cross-section of the tube,and most preferably, the exit opening is the cross-section of the tube.

This shear-force generator is arranged within a vessel containing aliquid preparation of prion protein, as rotation of the rotary elementin addition to exerting a shear-force of one pre-set intensity, which iscontrolled to a narrow range, generates a suction at the end which isprotruded by the encircling tube section such that all volume elementsof the liquid are moved through the space between the rotary element andthe encircling tube section, where the volume elements are consecutivelysubjected to the shear force.

The rotary element is arranged in a bearing, which is preferably coaxialto the tube and to the rotary element, e.g. the bearing can be arrangedin a tube section adjacent the at least one exit opening and oppositethe section encircling the rotary element. Preferably, the bearingcomprises or consists of a low-friction polymer tube, e.g. polytetrafluoro ethylene (PTFE), optionally having at least 2 or at least 4longitudinal convex or concave folds, arranged in a tube sectionadjacent the exit openings, between the rotary element and the tubeencircling it. The low-friction polymer tube of the bearing preferablyis arranged between one circumferential shoulder extending from therotary element and one circumferential shoulder protruding from theinner surface of the tube, e.g. one shoulder at one of the opposite endsof the bearing.

The shear force is controlled and set to a specific value by controllingthe rotation rate of the rotary element, preferably by controlling thedrive motor coupled to the rotary element.

Preferably, the shear force generator is controlled to one pre-setshear-force intensity corresponding to a rotation rate between 10 and10.000 Hz, preferably between 50 and 5000 or up to 2000 or 1000 Hz,precisely controlled to a range of maximally 1% of the rotation rateset, more preferably to a rotation rate with range of maximally +/−2 Hz,more preferably of maximally +/−1 Hz, with an outer diameter of therotary element of 1.95-2.05 mm arranged in a tube section with an innerdiameter of 1.55-2.75 mm, wherein the rotary element is a cylinder,optionally having a square flat end section.

In the alternative, the shear-force having a controlled shear-forceintensity can be generated by a shear-force generator which is asonicator, also referred to as an ultrasound emitter, which has a vesselfor receiving the liquid preparation containing the mixture to besubjected to shear-force, the vessel having an inner volume whichextends for a volume element only that is arranged in the distance fromthe sonicator surface (or sonotrode surface) and is parallel to thesurface section only, in which at least 75% to 90%, preferably at least95%, more preferably at least 99% of the maximum amplitude ispositioned. This volume element is e.g. arranged within the distance of0.5 mm to 50 mm from the surface of the sonicator and extends inparallel to the center of the surface fraction between vibration nodesof the sonicator surface, e.g. for maximally 10%, preferably formaximally 2% or 1% of the area between vibration nodes. The position ofthe volume element can e.g. be pre-determined by calculation of thesurface fraction of the sonotrode surface and the distance from thesonotrode surface in which maximum amplitude, and hence maximumshear-force is generated. Due to the specific arrangement of the volumeelement in the maximum vibration intensity, the liquid compositiontherein is subjected to a shear-force having an intensity of the limitedintensity range. For an efficient transfer of vibrations from thesonotrode surface to the volume element, the vessel preferably consistsof a material that is permissive to ultrasound, e.g. of polypropylene,poly tetrafluoro ethylene (PTFE) or other types of teflon, and atransfer liquid, e.g. water, is arranged between the vessel and thesonotrode surface. Preferably, the sonotrode forms one wall of atransfer liquid bath, e.g. a side wall and preferably the bottom wall,and the height of the transfer liquid in perpendicular to the sonotrodesurface is set to one wave-length of the ultrasound, e.g. at theresonance frequency of the sonotrode, or to an integral multiple of thewave-length of the ultrasound, e.g. at the resonance frequency of thesonotrode. The transfer liquid bath can be adapted or designed topre-set the height of the transfer liquid, the height being determinedin perpendicular to the sonotrode surface.

Preferably, the devices are arranged in an array of two or more devices,e.g. 7, 8, 12, 14 or 21 devices, arranged with their longitudinal axesvertically in a temperature-controlled housing. All devices of the arraycan be connected to and controlled by one computer, which is providedfor controlling the rotation rate of each rotary element individually.This array of devices is advantageous for treating aliquots of oneliquid composition of prion protein in parallel, i.e. without variationof the composition or state of the liquid composition, as e.g. couldoccur in subsequent treatment processes during storage of aliquots andday-to-day variations of treatment conditions. Using the array ofdevices, the process of the invention comprises the treatment ofaliquots of one liquid composition comprising the mixture of nativeconformation prion protein and seed in separate single devicesconcurrently, preferably with differing shear-forces each, which aregenerated by different rates of rotation applied to the rotary elements.

Generally, the device contains a control unit which is set forcontrolling the shear-force generator to at least one pre-set shearforce intensity to a range limited to a maximum of 10% of oneshear-force value, e.g. of the maximum shear-force, e.g. by setting orcontrolling a drive contained in or coupled to the shear-force generatorto a maximum range of 10% of one frequency, e.g. to a maximum of 10%,preferably of 1% or 0.1% of one frequency, e.g. of a pre-determinedfrequency or of the resonance frequency of the shear-force generator.The control unit can be a computer separate from the computercontrolling the shear-force generators or can be contained in the samecomputer. Preferably, both the control unit and the computer controllingthe shear-force generators are set up to store the shear-forceintensities for each shear-force generator and are preferably set up toadditionally store measuring results representing the content ofaggregated conformation prion protein, each in relation to theshear-force intensities. The measuring results representing the contentof aggregated conformation prion protein can be optical measurementresults from protein size separation, e.g. by chromatographic methodslike size exclusion chromatography, or electrophoresis, e.g. SDS-PAGE,optionally from Western blots for the admixture for optical density ofspecific bands, or optical measurement results for luminescence, forabsorbance or for light scatter generated by an optical detectorarranged to receive irradiation from the inner volume of the containerfor the admixture.

The device comprises or has access to a databank containing data onamounts of aggregated conformation prion protein, which data arepre-determined for one specific shear-force intensity, preferably for atleast two different shear-force intensities for a reference sample froma specific source of known diagnosis. The device, e.g. its computer, isset up for comparing the data provided in the computer-based databank todata representing amounts of aggregated conformation prion proteingenerated from an admixture of sample and native conformation prionprotein, wherein the comparison of amounts of aggregated conformationprion protein is made for the same shear-force intensity.

Generally preferred, all admixtures containing native conformation prionprotein and aggregated conformation prion protein, e.g. reference sampleor sample to be analysed, are in the same aqueous buffer, e.g. the sameconcentration of the same salts at the same pH value and at the sametemperature for all measurements.

The native conformation prion protein can e.g. be produced by expressionin a microorganism or in a cultivated cell, or be chemicallysynthesized. Preferably, the native conformation prion protein isprovided in aqueous solution.

Following or during subjecting the mixture or aliquots thereof, thecontent or increase of aggregated conformation prion protein isdetected. Generally, it is preferred to detect the amount of aggregatedconformation prion protein using an optical detector, which can beseparate from the shear-force generator so that the container containingthe aggregated conformation prion protein is transferred from theshear-force generator to the detector, which can e.g. be a plate reader.In the alternative, the optical detector can be arranged at thecontainer while the container is arranged at the shear-force generator,e.g. for measurement during resting phases of application of shear-forceintensities. In addition or in the alternative, the aggregated stateprion protein obtained by the process can be detected, optionallyfollowing contacting with proteinase, by size separation, e.g.chromatographically or electrophoretically, optionally with antibodydetection, e.g. in a Western blot, and/or by structure sensitivespectroscopy, e.g. by infrared spectroscopy (IR), preferablyFourrier-transformed IR (FT-IR), by NMR, especially ¹³C-NMR, and/orfluorescence spectroscopy, preferably after addition of a fluorescencedye being specific for aggregated conformation prion protein. Thesedetection steps have the advantage of providing structure-relatedinformation on the aggregated state prion protein generated, especiallyfollowing contacting the aggregated conformation prion protein withproteinase due to differences in proteinase resistance of aggregatedconformation prion protein.

Preferably, detection is by measuring the change of fluorescence of afluorescent dye added to the mixture, the dye being specific foraggregated conformation prion protein. Exemplary dyes are thosementioned in Eckroat et al., quoted above, e.g. Thioflavin T, ThioflavinS, Congo Red, thiophene-based amyloid ligands like luminescentconjugated polythiophenes (LCP), polythiophene acetic acid (PTAA) andluminescent conjugated oligothiophenes (LCO), Pittsburgh compound B,Aminonaphthalene 2-Cyanoacrylate (ANCA) probes, pegylatedphenylbenzoxazole derivatives, Pinacyanol, Chrysamine G, and dyescontaining at least on of the following scaffolds: Chalcone, Flavone,Aurone, Stilbene, Diphenyl-1,2,4-oxadiazole, Diphenyl-1,3,4-oxadiazole,Benzothiazole, Benzooxazole, Benzofuran, Imidazopyridine, Benzimidazole,Quinoline, Naphthalene.

In the alternative, the native conformation prion protein can belabelled with a fluorescent dye, e.g. by binding of a fluorescent dye tothe native conformation prion protein directly or by an intermediatespacer, e.g. fluorophore derivatives that contain reactive chemicalgroups such as isothiocyanates (reactive to primary amines, e.g.Lysines), succinylimide esters (reactive towards amino groups to formamido bonds, e.g. N-terminal amino acids), maleimide (reactive to freesulfhydryl groups, e.g. cysteine). Such fluorophore derivatives includecyanines, flurescein, rhodamine, Alexa Fluors, Dylight fluors, ATTO-TecDyes, BODIPY Dyes, SETA Dyes, SeTau Dyes, DYOMICS Dyes.

Optionally, the process can comprise the additional step of contactingthe admixture following application of shear-force intensity with atleast one compound selected from Thioflavin T, Thioflavin S, Congo Red,thiophene-based amyloid ligands like luminescent conjugatedpolythiophenes (LCP), polythiophene acetic acid (PTAA) and luminescentconjugated oligothiophenes (LCO), which compounds can beconformation-specific for aggregated conformation prion protein. Thisoptional additional step allows for distinguishing aggregatedconformations of prion protein by measuring their fluorescence. As someof these compounds can interfere with the change in conformation fromnative to aggregated conformation, at least one of them is contactedwith the admixture only subsequent to application of the shear-forceintensity. The addition of at least one of these compounds and theresultant fluorescence measurement is stored in the databank.

In the process comprising detection of aggregated conformation prionprotein, the device according to the invention can be used as a detectorfor the presence of seed in a sample. The device comprises at least onecontainer for receiving the admixture comprising the sample, thecontainer being coupled to or comprising a shear-force generator,preferably at least two containers with one shear-force generator foreach container. For detection, each container is provided with anirradiation source and an optical detector, preferably a fluorescencedetector, both directed at the container provided for receiving themixture of seed and native conformation prion protein. For example, thedevice comprises at least one, preferably at least two containers forreceiving a mixture of seed and native conformation prion protein, eachcontainer provided with a shear-force generator that is controlled togenerate at least one, optionally sequentially at least two differentshear-forces, and is provided with an optical detector arranged forreceiving light emitted from the container and an irradiation sourcearranged for irradiating the inner volume of the container. Generallypreferred, the containers are sealed. Preferably, the irradiationsource, e.g. a laser generator, is arranged with its beam path at anangle of 0° in the case of absorbance detector or in an angle equal toor below 180°, e.g. between 160° and 10° to the light path directed tothe detector, e.g. in the case of a fluorescence or scatter lightdetector.

Alternatively, the device comprises at least two shear force generators,and irradiation source and an optical detector, both directed at acontainer provided for receiving the mixture of seed and nativeconformation prion protein. Preferably, the irradiation source, e.g. alaser generator, is arranged with its beam path at an angle of 0° or atan angle of 180° or below 180°, e.g. between 160° and 10° to the lightpath directed to the optical detector.

Generally, the optical detector can be a fluorescence detector or ascatter light detector or an absorbance detector.

Preferably, the optical detector is coupled to a computer having accessto a databank that contains data on pre-determined amounts which arespecific for a single shear-force intensity, preferably for at least twodifferent shear-force intensities and specific for a seed from aspecific source are contained. These data are provided in a databank ona data carrier, especially in a computer-based databank, and thecomparison is carried out by a computer. Preferably, the databankcontains the medical diagnosis for a specific neurodegenerative diseaseand/or a subtype thereof in association with the pre-determined data onamounts of aggregated conformation prion protein, each amount generatedat one of at least two different shear-force intensities for each nativeconformation prion protein added and the diagnosis associated with thesample, and preferably additionally the specific source or sample towhich the diagnosis pertains. As for some neurodegenerative diseases,the rate of amplification of aggregated conformation prion proteinand/or the amount of aggregated conformation prion protein can differ independence on the point in time of the sample being taken during thedisease progression, it is preferred that the databank containsinformation on the age of disease onset, sex of the mammal, duration ofthe disease, progression and/or severity of the disease, preferably atleast data on the disease, subtype of the disease and severity of thedisease. This embodiment can be used for generating a prediction orprognosis of the disease progression rate, based on the combination ofseed-typing and seed-quantification in combination with other patientdata. Accordingly, this information is set up in the databank in anarrangement according to this information.

The invention also relates to the databank, preferably computer-based,containing the medical diagnosis for a specific neurodegenerativedisease and/or a subtype thereof in association with the pre-determineddata on amounts of aggregated conformation prion protein, data on eachamount generated at one of at least two different shear-forceintensities for each native conformation prion protein added to thesample and the diagnosis associated with the sample, and preferablyadditionally data on the specific source or on the sample to which thediagnosis pertains, e.g. kind of sample material, storage conditions,solutions contacting the sample. As for some neurodegenerative diseases,the rate of amplification of aggregated conformation prion proteinand/or the amount of aggregated conformation prion protein can differ independence on the point in time of the sample being taken during thedisease progression, it is preferred that the databank containsinformation on the age of disease onset, sex of the mammal, duration ofthe disease, progression and/or severity of the disease, preferably atleast data on the disease, subtype of the disease and severity of thedisease. Further preferred, the databank contains data on the type ofshear-force generator, on the temperature during application ofshear-force, on the detector used for detecting the amount of aggregatedprion protein. The databank, also described herein as a ReferenceInformation Database, due to containing data on the amounts ofaggregated prion protein generated for reference samples, for a sampleallows the identification of a diagnosis by matching the amounts ofaggregated conformation prion protein generated at at least one specificshear-force intensity to the data of the databank. As the presence of aspecific control or standard substance during generation of data on theamounts of aggregated prion protein from a sample or reference sample isoptional but not necessary, the databank has the advantage that the datacontained therein preferably are absolute data, i.e. the data areindependent from a specific control or standard substance. Accordingly,the process of the invention, especially when using the databank, alsogenerates absolute results.

Preferably, the databank comprises data obtained by secondary analysisof aggregated conformation prion protein obtained by the application ofone shear-force intensity by at least one of the following: sizeseparation, e.g. chromatographically or electrophoretically, optionallywith antibody detection, e.g. in a Western blot, structure sensitivespectroscopy, e.g. by infrared spectroscopy (IR), preferablyFourrier-transformed IR (FT-IR), NMR, especially ¹³C-NMR, and/orfluorescence spectroscopy, preferably after addition of a fluorescencedye being specific for aggregated conformation prion protein.Optionally, prior to the secondary analysis, the aggregated state prionprotein obtained by the process can be contacted with proteinase. Inthis embodiment, the databank has the advantage of containingstructure-related information on the aggregated state prion proteingenerated.

In a preferred embodiment, the databank comprises or is present incombination with the reference samples and/or aggregated conformationprion protein generated from the reference sample, preferably generatedat one shear-force intensity, optionally at least two aggregatedconformation prion proteins, each generated at one shear-forceintensity. Preferably, the aggregated conformation prion protein wasgenerated from the reference sample in at least two separate admixtures,each containing a different native conformation prion protein, e.g. oneof Aβ, Tau and α-Synuclein. In this embodiment, the databank is suitablefor a process for analysing or screening at least one compound for itseffect on the generation of aggregated conformation prion protein froman admixture containing the compound, at least one native conformationprion protein and the reference sample or aggregated conformation prionprotein generated at one shear-force intensity, acting as a seed.

The analytical process comprises the step of comparing the content ofaggregated conformation prion protein generated by the differentshear-forces to predetermined data on the content of aggregatedconformation prion protein produced by subjecting the nativeconformation prion protein in a mixture with a seed to the sameshear-forces. Therein, the predetermined data are contained in thedatabank described. The process for analysis for the presence ofdisease-related aggregated conformation prion protein in a biopsiedmammalian sample, which preferably is a process for a differential invitro analysis for identification and/or classification of aprion-related disease, comprises the steps of a) adding to the sample atleast one native conformation prion protein, b) subjecting the mixturecomprising the sample and the at least one native conformation prionprotein obtained in step a) to at least one shear-force intensity thatis controlled to have a uniform intensity having an intensity range ofmaximally 20% of one shear-force value for a pre-determined number ofcycles of a pre-determined time of shear-force acting and apre-determined resting phase, and c) following step b) determining thecontent of aggregated conformation prion protein for each of theshear-force intensities, comprising the step of d) comparing the contentof aggregated conformation prion protein determined in step c) topre-determined data on the content of aggregated conformation prionprotein, which content was determined for native conformation prionprotein in admixture with a reference sample subjected to the sameshear-force intensity as in step b), wherein these data are provided ina databank which in association with these data contains theneurodegenerative disease diagnosis for the patient from which thereference sample originates. Therein, step d) of comparing the contentof aggregated conformation prion protein determined for the biopsiedmammalian sample in admixture with at least one native conformationprion protein to the data on the content of aggregated conformationprion protein pre-determined for a reference sample in admixture with atleast one native conformation prion protein, each at the same controlledshear-force value, allows to assign the diagnosis associated with thepre-determined data to the biopsied mammalian sample.

Optionally, both for the sample and for the pre-determined data, thecontent of aggregated conformation prion protein generated by theapplication of one shear-force intensity can be determined as the rateof formation of aggregated conformation prion protein. This rate ofgenerating aggregated conformation prion protein by the application ofone shear-force intensity can e.g. be determined by measuring aggregatedconformation prion protein following the resting phase of each cycleand/or by continuously measuring aggregated conformation prion proteinduring application of the shear-force. Accordingly, the detector ispreferably coupled to a computer which is set up for determination ofthe rate of generation of aggregated conformation prion protein frommeasurement signals. Also in this embodiment the databank preferably isset up for storing pre-determined data on the rate of amplification ofthe aggregated conformation prion protein in dependence on theshear-force intensity applied and for storing in relation thereto dataon the diagnosis given for the patient from whom the sample originatesthat is used for the pre-determination.

The rate of formation of aggregated conformation prion protein, theoriginal content of aggregated conformation prion protein in the sampleand/or a rate of dissociation of the aggregated conformation prionprotein can be determined, especially separately for each shear-forceintensity.

The formation of one aggregated conformation prion protein at onespecific shear-force intensity is determined by the initialconcentration of aggregated conformation prion protein (p_(t=0)), thekinetic rate of native conformation prion protein incorporation into theaggregated conformation prion protein (k₊) at one specific shear-force,the kinetic rate of native prion protein dissociation (k_(d)) from theaggregated conformation prion protein, and by the initial concentrationof native conformation prion protein present in the admixture (m_(t=0)).The total amount of the detection signal determined for the content ofaggregated stated prion protein (F), e.g. total fluorescence, isdetermined by the specific detection signal of the aggregatedconformation prion protein (f_(s)). The parameter p_(t=0) is determinedby the original amount of one aggregated conformation prion proteinpresent in a mammalian sample and by the mixing ratio of the patientsample with the solution of native conformation prion protein. Theparameters k₊, k_(d) and f_(s) are chemical properties of the aggregatedconformation prion protein. The parameter m_(t=0) is pre-determined bythe specific assays conditions.

The change of one aggregated conformation prion protein content (dp)over time (dt) at one specific shear-force intensity and at one specifictime-point is determined by these parameters and by the presentconcentration of aggregated conformation prion protein (p) and thepresent concentration of native conformation prion protein (m). This issummarized by equation 1 (Eq. 1):dp/dt=k ₊ ·p·m−k _(d) ·p  Eq. 1

For example, the change of Thioflavin T Fluorescence (dF_(sim)) overtime (dt) at one specific shear-force intensity and at one specifictime-point is determined by the above mentioned parameters and by thepresent concentration of aggregated conformation prion protein (p) andthe present concentration of native conformation prion protein (m). Thisis summarized by equations 2 and 3 (Eq. 2, Eq. 3):dF _(R) /dt=f _(s)·(dp/dt)  Eq. 2dF _(R) /dt=f _(s)·(k ₊ ·p·m−k _(d) ·p)  Eq. 3

Parameters p_(t=0), k₊, k_(d) and f_(s) are independent variables whichare used to approximate the observed signal for the content ofaggregated conformation prion protein, e.g. the amount of fluorescence,between the time of the start of the reaction and the time of maximalfluorescence (F_(obs)(t)) by a non-linear regression analysis (R) usingnumerical or explicit solutions of Eq. 3. The nonlinear regressionanalysis minimizes the deviation of the approximated fluorescence(F_(R)(t)) from the observed fluorescence (F_(obs)(t)). The results ofthe regression analysis are approximated values for the parametersp_(t=0), k₊, k_(d) and f_(s), wherein k₊ is specific for one shear-forceintensity. Preferably, the values obtained for p_(t=0), k₊, k_(d) andf_(s) can be associated to specific disease conditions in the databank,and they can e.g. be used to discriminate healthy from diseased people.Preferably, the values p_(t=0), k₊, k_(d) and f_(s) which are determinedfrom the observed time-resolved signal for the content of aggregatedconformation prion protein at at least one shear-force intensity arepart of the determined content of aggregated conformation prion proteinfor each of the shear-force intensities for the mixture of the sampleand the at least one native conformation prion protein and for thepre-determined data of the mixture of the reference sample and the atleast one native conformation prion protein. Accordingly, in the processthe content of aggregated conformation prion protein is preferablydetermined in a time-dependent manner during subjecting the mixture toone shear-force intensity, e.g. it is determined as the time-resolvedcontent, and that the rate of formation of aggregated conformation prionprotein is determined by non-linear regression analysis from anapproximation on the determined time-resolved content of aggregatedconformation prion protein for each of the shear-force intensities.

Exemplary prion proteins are the proteins which in their aggregatedstate conformation cause or are present in the following diseases, withpreferred amino acid sequences of the prion protein indicated: Scrapiein sheep (cellular prion protein PrP^(c), major prion protein,accessible at http://www.uniprot.org/uniprot/P23907), bovine spongiformencephalitis in cattle (cellular prion protein PrP^(c), major prionprotein, accessible at http://www.uniprot.org/uniprot/P10279), chronicwasting disease in deer and elk (cellular prion protein PrP^(c), majorprion proteins, accessible at black deer:http://www.uniprot.org/uniprot/P47852, red deer:http://www.uniprot.org/uniprot/P67987, alpine musk deer:http://www.uniprot.org/uniprot/Q68G95), and Creutzfeld-Jacob disease,Gerstmann-Sträussler-Scheinker syndrome (cellular prion protein PrP^(c),including mutant proteins thereof), fatal familial insomnia in humans(prion protein), wherein prion disease in humans, including CreutzfeldJacob disease, Gerstmann-Sträussler-Scheinker (GSD) Syndrome, fatalfamilial insomnia (FFI) (major prion protein, accessible athttp://www.uniprot.org/uniprot/P04156), GSD and FFI are exclusivelyassociated with familial variants, CJD can be associated with familialvariants, Alzheimer disease (amyloid beta, Aβ, especially Aβ of 40(Aβ40) or 42 (Aβ42) amino acids, including mutant proteins thereof),especially Alzheimer disease or cerebral amyloid angiopathy in humans(beta amyloid (Aβ) A4 protein, accessible athttp://www.uniprot.org/uniprot/P05067, especially the partial sequencesbeta-amyloid protein 42, and beta-amyloid protein 40, and also familialvariants of the A4 protein), Alzheimer disease (tau and/or α-synucleinof human, mouse or rat, e.g. human alpha-synuclein accessible athttp://www.uniprot.org/uniprot/P37840, mouse alpha-synuclein accessibleat http://www.uniprot.org/uniprot/O55042 rat alpha-synuclein accessibleat http://www.uniprot.org/uniprot/P37377, human tau, accessible athttp://www.uniprot.org/uniprot/P10636 and corresponding tau sequences ofmice and rats, accessible at http://www.uniprot.org/uniprot/P35637),Alzheimer in mouse or rat (mouse Aβ accessible athttp://www.uniprot.org/uniprot/P12023; rat Aβ accessible athttp://www.uniprot.org/uniprot/P08592, and tau accessible athttp://www.uniprot.org/uniprot/P35637 and mouse α-synuclein, accessibleat http://www.uniprot.org/uniprot/O55042, rat alpha-synuclein accessibleat http://www.uniprot.org/uniprot/P37377), Parkinson (alpha-synuclein)and α-synucleopathies in humans (human alpha-synuclein accessible athttp://www.uniprot.org/uniprot/P37840), Parkinson disease andα-synucleopathies in murine disease models (alpha-synuclein accessibleat http://www.uniprot.org/uniprot/O55042) and Parkinson disease andsynucleopathies in rat disease models (alpha-synuclein accessible athttp://www.uniprot.org/uniprot/P37377), frontotemporal lobar dementia(TDP-43, accessible at http://www.uniprot.org/uniprot/Q13148),frontotemporal lobar dementia (tau, accessible athttp://www.uniprot.org/uniprot/P10636), and corresponding tau sequencesof mice and rats, frontotemporal lobar dementia (FUS, accessible athttp://www.uniprot.org/uniprot/P35637), and corresponding FUS sequencesof mice and rats, Amyotrophic Lateral Sclerosis (SOD1, accessible athttp://www.uniprot.org/uniprot/P00441), Amyotrophic Lateral Sclerosis(TDP-43, accessible at http://www.uniprot.org/uniprot/Q13148), diabetesmellitus type 2 (amylin, accessible athttp://www.uniprot.org/uniprot/P10997),), chorea Huntington (humanhuntingtin accessible at http://www.uniprot.org/uniprot/P42858,especially containing poly-Q expansions between amino acid sequenceposition 18 and 38), medullary carcinoma of the thyroid (calcitonin,accessible at http://www.uniprot.org/uniprot/P01258), cardiacarrhythmias, isolated atrial amyloidosis (atrial natriuretic factor,accessible at http://www.uniprot.org/uniprot/P01160), atherosclerosis(apolipoprotein A, accessible at http://www.uniprot.org/uniprot/P02647),rheumatoid arthritis (serum amyloid A, accessible athttp://www.uniprot.org/uniprot/P0DJI8), aortic medial amyloid (medin,accessible at http://www.uniprot.org/uniprot/Q08431), prolactinomas(prolactin), familial amyloid polyneuropathy (transthyretin, accessibleat http://www.uniprot.org/uniprot/P02766), hereditary non-neuropathicsystemic amyloidosis (lysozyme, accessible athttp://www.uniprot.org/uniprot/P61626), dialysis related amyloidosis(beta-2-microglobulin, accessible athttp://www.uniprot.org/uniprot/P61769), Finnish amyloidosis (gelsolin,accessible at http://www.uniprot.org/uniprot/P06396), lattice cornealdystrophy (keratoepithelin, accessible athttp://www.uniprot.org/uniprot/Q15582), cerebral amyloid angiopathy(beta-amyloid), also of the Icelandic type (cystatin, accessible athttp://www.uniprot.org/uniprot/P01034), systemic AL amyloidosis(immunoglobulin light chain AL), sporadic inclusion body myositis(S-IBM), tauopathies involving the agglomeration of tau protein (humantau-protein accessible at http://www.uniprot.org/uniprot/P10636),Tauopathies involving the agglomeration of tau protein in murine diseasemodels (mouse tau-protein accessible athttp://www.uniprot.org/uniprot/P10637), Tauopathies involving theagglomeration of tau protein in rat disease models (rat tau-proteinaccessible at http://www.uniprot.org/uniprot/P19332)). Herein, tauprotein includes isoforms, especially isoforms as described by Barghornet al. (loc. cit.), human tau23 of 352 amino acids, lacking N-terminalinserts I1 and I2 between E45 and A103 and lacking R2 between V275 andV306, human tau37 of 381 amino acids, lacking N-terminal insert I2between D74 and A103 and lacking R2 between V275 and V306, human tau39of 410 amino acids, lacking R2 between V275 and V306, human tau24 of 383amino acids, lacking N-terminal inserts I1 and I2 between E45 and A103,human tau34 of 412 amino acids, lacking N-terminal insert I2 between D74and A103, human tau K19 of 98 amino acids containing amino acids Q244 toE372 but lacking R2 between V275 and V306, human tau K18 containingamino acids Q244 to E372, each in comparison to human tau40 of 441 aminoacids (human tau isoform F accessible athttp://www.uniprot.org/uniprot/P10636#P10636-8).

The amino acid sequences are accessible in protein databanks, e.g. inUniprot. The amino acid sequence of the prion protein used in theprocess of the invention can optionally have an added synthetic ornatural amino acid section, e.g. a detectable tag.

A preferred device for use in the process comprises at least one,preferably at least two separate shear-force generators, each controlledto apply one shear-force intensity to the admixture of sample and nativeconformation prion protein.

As the databank contains the amounts of aggregated conformation prionprotein produced from reference samples for at least two specificshear-force intensities in association with at least the disease, theprocess can optionally be carried out without a positive controlcontaining a known type and/or a known amount of aggregated conformationprion protein, and/or without a negative control containing no seed,i.e. native conformation prion protein only.

The invention is now described in greater detail by way of examples withreference to the figures that show in

FIGS. 1 to 6 fluorescence measurements specific for the amplification ofthe aggregated conformation of prion protein at different shear-forceintensities for samples of known diagnosis,

FIGS. 7-10 fluorescence measurements specific for the amplification ofthe aggregated conformation of prion protein at different shear-forceintensities for samples from post mortem brain samples,

FIG. 11 a schematic overview of an embodiment of the process,

FIG. 12 a schematic overview of an embodiment of the process,

FIG. 13 a schematic cross-section of a preferred device for use in theprocess,

FIG. 14 an exploded schematic view of the device of FIG. 13,

FIG. 15 a schematic view of a device,

FIG. 16 a schematic view of an embodiment of the device,

FIG. 17 a schematic cross-section along line A1 of FIG. 16, and

FIG. 18 a schematic cross-section along line A2 of FIG. 17.

Example: Amplification of Aggregated State Conformation at DifferentShear-Force Intensities

As samples, a 1:50 volume portion of brain homogenate was used and mixedwith 2 mg/ml alpha-synuclein as the native conformation prion protein.The brain homogenate was prepared by homogenization in coldphosphate-buffered saline (PBS) containing 0.5% Triton X-100 and 1×complete EDTA-free protease inhibitor cocktail (Roche, Cat. No.11873580001) using 20 strokes with a dounce homogenizer on ice. Brainsamples were post-mortem from one healthy control (NEG.) and fourdifferent synucleopathy disease patients: Idiopathic Parkinson's Disease(IPD), Parkinson's Disease with Dementia (PDD), Dementia withLewy-Bodies (DLB), Muliple System Atrophy (MSA). The suspension wasclarified by centrifugation at 2000×g for 45 s, and the supernatant wasused.

As another sample, 360 μl human cerebrospinal fluid (CSF), stored at−80° C. were mixed with 40 μl cold 10×PBS containing 0.5% Triton X-100and 1× complete EDTA-free protease inhibitor cocktail and clarified bycentrifugation at 2000×g for 45 s, giving the supernatant as humancerebrospinal fluid extract (CSFE). In this example the CSF sample waspost-mortem from the same patient of Parkinson's disease with dementia(PDD) that was used above.

The native conformation prion protein was recombinantly expressed humanalpha-synuclein of approx. 5.0 mg/ml water, stored at −80° C. and thawedat 37° C. at very mild agitation. The protein concentration was adjustedto 2.22 mg/ml using sterile-filtered water and 13.5 ml thereof weremixed with 1.5 ml of 10× concentrated PBS to yield 2.0 mg/ml humanalpha-synuclein in PBS.

On ice, 15 ml of 2.0 mg/ml human alpha-synuclein in PBS were combinedwith 300 μl brain homogenate supernatant, or alternatively with 300 μlCSFE. Of this mixture, twelve identical aliquots of 1.2 ml each werefilled into 1.5 ml sealed polypropylene test tubes (SureLock,Eppendorf). Into each test tube, one rotary shearing device having arotor of 2.00 mm within a tube at a gap width of 0.30 mm was inserted.These assemblies were incubated at 37° C. for 15 min. Shear-force wasapplied by rotating the rotors with control of the rotating rate to atmaximum 1% from the rate set for 5 s with a subsequent resting phase of295 s for a total of 22 h. The rotating rates used are indicated inFIGS. 1 to 9. Generally, the device corresponded to WO 2012/110570.

Samples of 20 μl were taken from each reaction mixture at time 0 h, 3 h,6 h, 9 h, 12 h, and at 22 h under shear-force. The amplification ofaggregated state conformation was determined by fluorescencespectroscopy of 15 μl of the sample taken after mixing with 135 μlThioflavin T stock solution (30 μM Thioflavin T in PBS buffer solution)with excitation at 450 nm (bandwidth 10 nm) and detection at 482 nm(bandwidth 20 nm).

The brain samples originated from patients diagnosed with the followingParkinson syndromes: Idiopathic Parkinson Disease (IPD), ICD-10: G20

-   -   Dementia in Parkinson's disease (PDD), ICD-10: G20, F02.3    -   Multisystem Atrophy (MSA), ICD-10: G90.3:    -   Dementia with Lewybodies (DLB), ICD-10: G31.8, F02.3    -   Healthy Control (NEG.),

and the CSF sample originated from a patient diagnosed with

-   -   Dementia in Parkinson's disease (PDD), ICD-10: G20, F02.3

For diagnosis, the ICD-10 codes (available at www.ICD-code.de, athttp://apps.who.int/classifications/icd10/browse/2010/en#, athttp://www.who.int/classifications/icd/en/, and athttp://www.who.int/classifications/icd/en/GRNBOOK.pdf) were used.

In detail, F00 Dementia in Alzheimer's disease: Alzheimer disease (AD)is a primary degenerative cerebral disease of unknown etiology withcharacteristic neuropathological and neurochemical features. Thedisorder is usually insidious in onset and develops slowly but steadilyover a period of several years. Associated with the deposition of seedof abeta protein, tau protein, and sometimes synuclein protein

-   -   F00.0*, G30.0*: Dementia in Alzheimer's disease with early        onset. Dementia in Alzheimer disease with onset before the age        of 65, with a relatively rapid deteriorating course and with        marked multiple disorders of the higher cortical functions.        Includes: (i) Alzheimer disease, type 2; (ii) Presenile        dementia, Alzheimer type; (iii) Primary degenerative dementia of        the Alzheimer type, presenile onset.    -   F00.1*, G30.1*: Dementia in Alzheimer's disease with late onset.        Dementia in Alzheimer disease with onset after the age of 65,        usually in the late 70s or thereafter, with a slow progression,        and with memory impairment as the principal feature.        Includes (i) Alzheimer disease, type 1; (ii) Primary        degenerative dementia of the Alzheimer type, senile onset; (iii)        Senile dementia, Alzheimer type.    -   F00.2*, G30.8*: Dementia in Alzheimer's disease, atypical or        mixed type. Atypical dementia, Alzheimer type    -   F00.8, G30.9: Dementia in Alzheimer's disease, unspecified

F02 Dementia in other diseases classified elsewhere. Cases of dementiadue, or presumed to be due, to causes other than Alzheimer disease orcerebrovascular disease. Onset may be at any time in life.

-   -   F02.0*, G31.0*: Dementia in Pick's disease (Frontotemporal        lobular Dementia, FTD). A progressive dementia, commencing in        middle age, characterized by early, slowly progressing changes        of character and social deterioration, followed by impairment of        intellect, memory, and language functions, with apathy, euphoria        and, occasionally, extrapyramidal phenomena.    -   F02.2*, G10*: Dementia in Huntington's disease (HD). A dementia        occurring as part of a widespread degeneration of the brain. The        disorder is transmitted by a single autosomal dominant gene.        Symptoms typically emerge in the third and fourth decade.        Progression is slow, leading to death usually within 10 to 15        years. Includes: Dementia in Huntington chorea    -   F02.3*, G20*: Dementia in Parkinson's disease (PDD): dementia        developing in the course of established Parkinson disease. No        particular distinguishing clinical features have yet been        demonstrated. Includes (i) Hemiparkinsonism, (ii) Paralysis        agitans, (iii) Parkinsonism or Parkinson disease (NOS (not        otherwise specified), idiopathic, primary)    -   F02.3*, G31.82: Lewy body Dementia (DLB), Lewy Body Disease        (LBD). A progressive degenerative dementia. Persons with LBD        will show markedly fluctuating cognition. Persistent or        recurring visual hallucinations with vivid and detailed pictures        are often an early diagnostic symptom.

A81 Atypical virus infections of central nervous system. Prion diseasesof the central nervous system.

-   -   A81.0*, F02.1*. Dementia in Creutzfeldt-Jakob disease. A        progressive dementia with extensive neurological signs, due to        specific neuropathological changes that are presumed to be        caused by a transmissible agent. Onset is usually in middle or        later life, but may be at any adult age. The course is subacute,        leading to death within one to two years.    -   A81.8: Other atypical virus infections of central nervous        system: Kuru    -   A81.9: Atypical virus infection of central nervous system,        unspecified: Prion disease of central nervous system.

Amyloidosis:

-   -   168.0* Cerebral amyloid angiopathy (E85.−+)    -   [Possibly to be extended]

Parkinson Syndromes:

-   -   G20: Idiopathic Parkinson Disease (IPD)    -   G20, F02.3: Dementia in Parkinson's disease (PDD)    -   G90.3: Multisystem Atrophy (MSA)    -   G31.8, F02.3: Dementia with Lewybodies (DLB)

Motor Neuron Disease:

G12.2: Motor neuron disease: includes (i) Familial motor neuron diseaseand (ii) Amyotrophic Lateral sclerosis (ALS).

FIGS. 1-6 show the fluorescence detected in 3 independent repetitions ofthe examples. In FIG. 1, the sample was BN449-PDD, in FIG. 2 BN379-IPD,in FIG. 3 BN175-MSA, in FIG. 4 BN526-DLB, in FIG. 5 BN449-PDD total CSF,and in FIG. 6 BN276 Healthy Control.

The results show that in the healthy control (NEG.), no aggregatedconformation prion protein was generated. All the samples from thepatients diagnosed with Parkinson syndromes resulted in the generationof amplification that was dependent on the shear-force intensity(rotation rate, application time, resting time and cycle number) anddependent on the origin of the sample. The process was highlyreproducible in three independent experiments performed on the samebrain tissues.

In FIGS. 1-10, the shear-force intensities are given as rpm of therotary shear-force generator on the X-axis, the amounts of aggregatedconformation prion protein detected are given on the Y-axis with theindividual curves for given for the time points indicated on the right(h application of shear-force intensities).

In FIGS. 1-4 and 6, the contents of aggregated conformation prionprotein generated at a pre-determined application of one shear-forceintensity each is depicted, showing a specific pattern of amplificationfor each sample for the shear-force intensities. In FIG. 5, the contentsof aggregated conformation prion protein at 0 h, and generated at 3 h, 9h and 12 h, respectively, are depicted, showing the different rates ofamplification at different shear-force intensities. For the process ofthe invention it is therefore generally preferred that the shear-forceintensity is pre-determined and the same for the sample and forpre-determined contents, e.g. the shear-force intensity can generally bepre-determined for shear-force applied, duration of shear-forceapplication, duration of resting phase for each cycle, and repetitionnumber of cycles.

The results show that the process of the invention differentiatesbetween samples of different pathologies and between subtypes, e.g.specific disease presentation of individual patients.

FIGS. 7-9 show results for the same process conditions for post mortembrain (BN) samples, wherein numbers designate individual samples. Incontrast to the samples of FIGS. 1-6, the disease status of patientsfrom whom the samples of FIGS. 7-9 originate is unknown to the personsinvolved in performing the analytical process. In these Figures, thecontent of aggregated prion protein generated at different time pointsis indicated.

The results depicted in FIGS. 7-9 show that the amplification greatlyvaries between samples and between points in time of shear-forceapplication.

FIG. 10 shows the amount of aggregated conformation prion protein froman admixture of a post mortem sample of a brain histologicallydetermined as Alzheimer and human α-synuclein as the native conformationprion protein. The result shows that the Alzheimer sample which wasdiagnosed to contain Aβ and tau protein aggregates when subjected tospecific shear-force intensities did not significantly induce thegeneration of aggregated conformation prion protein from the nativeconformation α-synuclein. This results demonstrates that at least forthis Alzheimer sample, generation of aggregated conformation prionprotein from a native conformation prion protein is specific for thesample.

A comparison of the amplification of aggregated prion protein at singleshear-force intensities, i.e. for different rotation rates at the samecycles for a total of 22 h allows to identify similar patterns ofamplification, preferably an identification of the unknown sampleaccording to similarities of the amplification pattern generated from asample of known diagnosis. In this process, the sample of knowndiagnosis serves as a reference sample. In detail, the samples of FIGS.8 and 9 show a similar amplification pattern at specific shear-forceintensities (22 h) as the IPD sample of FIG. 2.

Therefore, it is assumed that the process can differentiate samplesaccording to the progression of accumulation of aggregated prion proteinduring disease.

FIG. 11 schematically shows an overview of the process for analysis.Reference samples, e.g. post mortem brain tissue samples, serum, CSF orurine, each in association with the specific diagnosis for aneurodegenerative disease are provided as a library of reference seeds(Reference Seed Library). In the library, the reference samples can beassigned to the respective prion protein, represented by alpha-synuclein(α-Syn), tau (Tau) or amyloid beta (Aβ). For the reference samples,pre-determined data on the amounts of aggregated conformation prionprotein is generated by application of specific shear-force intensities(SSA) from an admixture containing reference sample and nativeconformation prion protein. The measurement can be by opticaldetermination of the amounts of aggregated conformation prion protein inWestern blots or using an optical detector receiving irradiation fromthe admixture. As indicated by the double arrows between the ReferenceSeed Library and the databank Reference Information Database, the dataon the detected amounts (Amplification Profiles) of aggregatedconformation prion protein in respect of each shear-force intensity arestored in a computer and stored in a databank (Reference Seed Library)in association with the respective specific diagnosis (Disease, IPD,PDD, MSA, DLB, FTD, AD), preferably including at least one subtype (G20,F02.3, G90.3, G31.82, G31.0, F00.0, G30.0, G30.1, G30.8, G30.9)according to a classification (ICD10). The databank (ReferenceInformation Database) therefore for each reference sample (SampleInformation) in association with the specific disease, preferably itssubtype, for the native conformation prion protein used (Aβ, Tau, α-Syn)contains the amounts of aggregated conformation prion protein for eachshear-force intensity (Amplification Profiles).

As indicated by the double arrows between the Diagnostic Process and theReference Seed Library, patient samples can be integrated into thereference samples (Reference Seed Library) once the diagnosis associatedwith the sample is known.

As generally preferred, the sample to be analysed (Patient Sample) inadmixture with the same native conformation prion protein (separatedadmixtures for each of α-Syn, Tau and Aβ) as at least one referencesample of the Reference Seed Library is subjected to the same at leastone shear-force intensity (SSA), and amounts of aggregated conformationprion protein are measured. Generally preferred, the sample to beanalysed is of the same type as the reference sample, e.g. blood serum,lymph fluid, urine, CSF or a tissue sample.

The amount of aggregated conformation prion protein generated atspecific shear-force intensities generated for a sample (Patient sample)is compared to the amount of aggregated conformation prion proteingenerated at the same shear-force intensities for the same nativeconformation prion protein (Aβ, Tau, α-Syn) each (Computer), allowingthe identification (Diagnosis) of the diagnosis associated to thereference sample in the databank (Reference Information Database) bythis comparison.

FIG. 12 in addition to FIG. 11 shows that preferably for eachapplication of a shear-force intensity in addition to a sample to beanalysed (Patient), native prion protein only, i.e. without a seed(negative Control) and a reference sample (Reference Seed) as a positivecontrol can optionally be used in the process in parallel to the sample.The reference sample can e.g. be taken from the Reference Seed Library.Generally, the reference sample can be an aggregated conformation prionprotein produced by application of one shear-force intensity to anadmixture of one patient sample of known disease with nativeconformation prion protein, preferably followed by at least one furtherapplication of the same shear-force intensity to an admixture of analiquot of the resultant product with the same native conformation prionprotein.

Further, FIG. 12 shows that preferably for each application of ashear-force intensity to a sample or reference sample, at least one ofthe following data is stored: the specific shear-force generator or itsdrive D, preferably including the frequency generated by its drive D,the type of lid L, the type of sample compartment S, e.g. comprising thelid L, the thermostat T and the optical detector O, the currenttemperature and/or the specific temperature control element T and/or thetype and/or specific optical detector O, including the opticalmeasurement data are stored, e.g. using an interface (ComputerInterface) coupled to each shear-force generator of the device andcoupled to the computer (Computer) for transmitting the data. As furtherpreferred, FIG. 12 shows that for measuring the amount of aggregatedconformation prion protein, the computer is set up to store thetemperature, time course (Timing, t), the shear-force generator (Drive,D), and the optical detector (Optics, O) for each application of ashear-force intensity, preferably including storing the time course ofthe detected amounts of aggregated conformation prion protein, e.g. inthe form of amplification profiles for each native conformation prionprotein in an admixture using e.g. a programme for storing these data(Device Software). Further, the device optionally comprises a aprogramme (Evaluation Software) for generating from these data which aremeasured and stored during the application of at least one shear-forceintensity the amounts of aggregated conformation prion protein, each forthe combination of specific native conformation prion protein of theadmixture (Aβ, Tau, α-Syn), with the measured values for shear-forceintensity, temperature, time-course of the application of shear-forceintensity, specific shear-force generator and/or specific optics, e. g.detector. The evaluation software has access to the databank. The devicecontains or has access to the databank comprising pre-determined amountsof aggregated conformation prion protein generated at at least oneshear-force intensity for reference samples (Reference InformationDatabase), and the computer is set up to compare the amplificationprofiles generated for samples for each shear-force intensity (doublearrows between Computer and Reference Information Database), allowingthe association of a disease, preferably including its subtype, storedfor a reference sample to the sample analysed. The computer isoptionally set up to edit this diagnosis (Diagnosis).

FIGS. 13 and 14 show cross-sections of a preferred shear-force generatorfor use in the invention. By way of a connection 1, e.g. a data transferline to a computer (not shown), a drive control unit 2 is controlled bythe computer. The drive control unit 2 controls the drive motor 3 to auniform rotation frequency. The drive control unit 2 and the drive motor3 can also be termed drive D. The rotor 9 is arranged on an axle 9 athat is connected to the drive motor 3 by a coupling 4, which preferablyis a magnetic coupling. The rotor 9 is arranged within a container 8 anda stator 10 is arranged between the rotor 9 and the container 8. Thestator 10 is arranged with a spacing to rotor 9, which preferably is auniform spacing to the radial outer surface of rotor 9, e.g. forming achannel of ring-shaped cross-section, and stator 10 is arranged with aspacing to container 8. Preferably, stator 10 is mounted on the lidsection L, also containing a bearing for axle 9 a. Optionally, lidsection L contains drive motor 3 and drive control unit 2. Stator 10preferably has extensions 10 e opposite the axle 9 a carrying rotor 9,which extensions 10 e form a funnel having an inlet 10 i and reducingthe free inner volume of container 8 in the region between the end ofrotor 9 opposite the axle 9 a and container 8. The inlet 10 i of thestator 10 is preferably arranged coaxially to rotor 10 and forms thenarrow exit of the funnel formed by extensions 10 e, guiding liquid tothe front surface section of rotor 9, allowing a pumping action by rotor9 to move liquid into the spacing between the radial surface of rotor 9and stator 10. Opposite the inlet 10 i, the outlet opening 10 o has atleast the cross-section of the spacing between rotor 9 and stator 10.

The container 8 provided for receiving a sample 20 is arranged within ahousing 7, which preferably at least sectionally is a thermostat T,preferably having form fit to the container 8. Preferably, thethermostat T for each container 8 has a temperature sensor and isindependently computer-controlled. The open end of the container 8 isclosed by a lid 6 and a seal 5. As shown, the section of housing 7embracing the section of container 8 between extensions 10 e of stator10 and the bottom of the container 8 opposite its opening is providedwith a light source 17 arranged to irradiate the inner volume of thecontainer 8 and an optical detector 14 arranged to receive irradiationexiting the inner volume of the container 8, wherein preferably the beampath of the light source 17 crosses the beam path of the opticaldetector 14 in the area between the inlet 10 i and the bottom of thecontainer 8. The beam path 19 generated by the light source 17 can crossthe exiting beam path 11 towards the optical detector 14 e.g. at anangle of 90°. Both the light source 17 and the optical detector 14 arecoupled to a computer for control of the light source 17 and forreceiving measurement signals from the detector 14. In the exiting beampath 11, a wavelength discriminator 13, e.g. an optical filter can bearranged. In the beam path 19 generated by the light source 17, awavelength discriminator 18, e.g. an optical filter can be arranged. Thehousing 12 for the detector 14 and/or for the light source 17 has adataline 16 for transmitting data on irradiation and measurement signalsto a computer. Optionally, a control unit 15 for controlling thethermostat T and/or the light source 17 is arranged at the housing 12.Preferably, housing 12 containing the light source 17, optionallyprovided with a wavelength discriminator 18, and optical detector 14,optionally provided with a wavelength discriminator 13, form anintegrated optical unit O. The optical unit O can be mounted releasablyto thermostat T and to adjacent lid section L, wherein these elementsform a recess for receiving a portion of container 8.

The scale indicated in FIGS. 13 and 14 is an exemplary scale, indicatingthat preferably each container 8 is provided with an individualcontrolled shear-force generator comprising a rotor 10 within a stator9, a thermostat T, a light source 17 and a detector 14 and a controlleddrive motor 3 within a scale of 7 to 12 mm, preferably 9 mm, forarrangement in a row or grid. FIG. 15 shows a generally preferredarrangement of at least two, e.g. of 8 or 12 containers that areconnected to one another, each provided with a separate shear-forcegenerator comprising a drive D and a lid section L, wherein drives D andlid sections L as well as thermostats T and optical units O,respectively are connected to one another in the same spacing forarrangement around spaced-apart coupled containers 8.

FIG. 16 shows another embodiment, wherein the shear-force generator isformed of a rotor 9 and stator 10 consisting of a wall section of thecontainer 8. The wall section of the container 8 forming the stator 10is e.g. arranged for a portion, e.g. at least 1° to 270°, e.g. for 30°to 180° about the circumference of the rotor 9 at a constant distance.As shown in FIG. 16, the rotor 9 is preferably arranged asymmetricallywithin container 8, leaving a free inner volume section for opticaldetection.

The container 8 can e.g. have a circular, oval or egg-shapedcross-section, and the rotor 9 can be arranged within a part of thecross-section having a smaller or larger diameter.

FIGS. 17 and 18 show that the container 8 preferably forms a stator 10arranged at a constant distance from the rotor 9 for a portion about thecircumference of the rotor 9, wherein this distance forms the smallestpassage between rotor 9 and stator 10. Accordingly, the surface of rotor9 preferably is parallel to the stator 10 for a portion of thecircumference of the rotor 9.

REFERENCE NUMERALS

 1 connection  2 drive control unit  3 drive motor  4 coupling  5 seal 6 lid  7 housing  8 container  9 rotor  9a axle 10 stator 10eextensions 10o outlet opening 10i inlet 11 exiting light path 12 housingcontaining optical detector and light source 13 wavelength discriminator14 optical detector 15 control unit 16 dataline 17 light source 18wavelength discriminator 19 beam path from light source 20 sample Ddrive L lid section T thermostat S sample compartment O optical unit

The invention claimed is:
 1. A process for analysis for the presence ofneurodegenerative prion-protein aggregation disease-related aggregatedconformation prion protein in a biopsied mammalian sample, the diseasebeing one of Idiopathic Parkinson's Disease (IPD), Parkinson's Diseasewith Dementia (PDD), Dementia with Lewy-Bodies (DLB) or Multiple SystemAtrophy (MSA), comprising the steps of a) adding alpha-synuclein as anative conformation prion protein to the sample to form a mixture andadding to the mixture at least one luminescent dye that is specific forthe aggregated conformation prion protein and measuring the luminescenceof the dye, b) subjecting the mixture comprising the sample andalpha-synuclein obtained in step a) to at least one shear-forceintensity that is computer-controlled to have a uniform intensity havingan intensity range of maximally 20% of one shear-force value for one ora plurality of cycles of shear-force acting and resting; c) followingstep b), determining via computer and storing the content of aggregatedconformation prion protein for each of the shear-force intensities, andd) comparing, with a computer, the content of aggregated conformationprion protein determined in step c) to data in a computer-based databankon the content of aggregated conformation prion protein, which contentwas determined for alpha-synuclein as a native conformation prionprotein subjected to the same shear-force intensity as in step b),wherein the data on the content of aggregated conformation prion proteinwas determined in alpha-synuclein as a native conformation prion proteinin admixture with a reference sample and these data are provided in thecomputer-based databank which in association with these data containsthe neurodegenerative prion-protein aggregation disease diagnosis of oneor more of Idiopathic Parkinson's Disease (IPD), Parkinson's Diseasewith Dementia (PDD), Dementia with Lewy-Bodies (DLB) and Multiple SystemAtrophy (MSA) for the patient from which the reference sampleoriginates.
 2. The process according to claim 1, wherein prior to stepb) the mixture is divided into aliquots and in step b) at least twoaliquots are subjected to a different shear-force intensity each and instep c) the content of aggregated conformation prion protein isdetermined for each aliquot and in step d) the content of aggregatedconformation prion protein determined in step c) for each aliquot iscompared to data on a content of aggregated conformation prion protein.3. The process according to claim 1, wherein in step b) the mixture issubjected to a succession of at least two different shear-forceintensities and the content of aggregated conformation prion protein isdetermined during or following subjecting the mixture to each one of theshear-force intensities.
 4. The process according to claim 1, comprisingirradiating the mixture with light having a wavelength for excitingluminescence in the dye and measuring the luminescence of the dye duringshear-force acting of step b) or during a resting phase of step b),without moving the volume occupied by the mixture relative a theshear-force generator generating the shear-force in step b).
 5. Theprocess according to claim 1, wherein in step b) the rate of formationof aggregated conformation prion protein is determined from the contentof aggregated state prion protein determined at the at least oneshear-force intensity and the data contain the rate of formation at thesame shear-force intensity.
 6. The process according to claim 1, whereinthe content of aggregated conformation prion protein is determined asthe time-resolved content and that the rate of formation of aggregatedconformation prion protein is determined by non-linear regressionanalysis of an approximation on the determined time-resolved content ofaggregated conformation prion protein for each of the shear-forceintensities.
 7. The process according to claim 1, characterized byadding at least one aggregated conformation prion protein to at leastone aliquot of the mixture comprising the sample and alpha-synuclein asa native conformation prion protein, wherein the alpha-synuclein inaggregated conformation is produced by subjecting native alpha-synucleinas a native conformation prion protein to a uniform shear-forcecontrolled to an intensity range of maximally 1% of one shear-forceintensity.
 8. A process for analysis for the presence ofneurodegenerative prion-protein aggregation disease-related aggregatedconformation prion protein in a biopsied mammalian sample, comprisingthe steps of a) adding alpha-synuclein as a native conformation prionprotein to the sample to form a mixture; b) subjecting the mixturecomprising the sample and alpha-synuclein as a native conformation prionprotein obtained in step a) to at least one shear-force intensity thatis computer-controlled to have a uniform intensity having an intensityrange of maximally 20% of one shear-force value for one or a pluralityof cycles of shear-force acting and resting; c) following step b),determining via computer and storing the content of aggregatedconformation prion protein for each of the shear-force intensities, andd) comparing, with a computer, the content of aggregated conformationprion protein determined in step c) to data in a computer-based databankon the content of aggregated conformation prion protein, which contentwas determined for alpha-synuclein as a native conformation prionprotein subjected to the same shear-force intensity as in step b),wherein the data on the content of aggregated conformation prion proteinwas determined in alpha-synuclein as a native conformation prion proteinin admixture with a reference sample and these data are provided in thecomputer-based databank which in association with these data containsthe neurodegenerative prion-protein aggregation disease diagnosis of oneor more of Idiopathic Parkinson's Disease (IPD), Parkinson's Diseasewith Dementia (PDD), Dementia with Lewy-Bodies (DLB) and Multiple SystemAtrophy (MSA) for the patient from which the reference sampleoriginates, the method further comprising irradiating the mixture withlight having a wavelength that is scattered by the aggregatedconformation prion protein and measuring scattered light exiting theadmixture during step b), or during a pause of step b), with or withoutmoving the volume occupied by the mixture relative a the shear-forcegenerator generating the shear force in step b).
 9. The processaccording to claim 8, comprising adding to the mixture at least oneluminescent dye that is specific for the aggregated conformation prionprotein prior to the step of subjecting the mixture to at least twodifferent shear-force intensities and measuring the luminescence of thedye.